Yonsei Med J.  2018 Nov;59(9):1096-1106. 10.3349/ymj.2018.59.9.1096.

Inhibition of miR-128 Abates Aβ-Mediated Cytotoxicity by Targeting PPAR-γ via NF-κB Inactivation in Primary Mouse Cortical Neurons and Neuro2a Cells

• Geng L
• Zhang T
• Liu W
• Chen Y

• KMID: 2422495
• DOI: http://doi.org/10.3349/ymj.2018.59.9.1096

Abstract

PURPOSE
Alzheimer's disease (AD) is the sixth most common cause of death in the United States. MicroRNAs have been identified as vital players in neurodegenerative diseases, including AD. microRNA-128 (miR-128) has been shown to be dysregulated in AD. This study aimed to explore the roles and molecular mechanisms of miR-128 in AD progression.
MATERIALS AND METHODS
Expression patterns of miR-128 and peroxisome proliferator-activated receptor gamma (PPAR-γ) messenger RNA in clinical samples and cells were measured using RT-qPCR assay. PPAR-γ protein levels were determined by Western blot assay. Cell viability was determined by MTT assay. Cell apoptotic rate was detected by flow cytometry via double-staining of Annexin V-FITC/PI. Caspase 3 and NF-κB activity was determined by a Caspase 3 Activity Assay Kit or NF-κB p65 Transcription Factor Assay Kit, respectively. Bioinformatics prediction and luciferase reporter assay were used to investigate interactions between miR-128 and PPAR-γ 3"²UTR.
RESULTS
MiR-128 expression was upregulated and PPAR-γ expression was downregulated in plasma from AD patients and amyloid-β (Aβ)-treated primary mouse cortical neurons (MCN) and Neuro2a (N2a) cells. Inhibition of miR-128 decreased Aβ-mediated cytotoxicity through inactivation of NF-κB in MCN and N2a cells. Moreover, PPAR-γ was a target of miR-128. PPAR-γ upregulation attenuated Aβ-mediated cytotoxicity by inactivating NF-κB in MCN and N2a cells. Furthermore, PPAR-γ downregulation was able to abolish the effect of anti-miR-128 on cytotoxicity and NF-κB activity in MCN and N2a cells.
CONCLUSION
MiR-128 inhibitor decreased Aβ-mediated cytotoxicity by upregulating PPAR-γ via inactivation of NF-κB in MCN and N2a cells, providing a new potential target in AD treatment.

Keyword

Alzheimer's disease; microRNA-128; PPAR-γ; NF-κB

MeSH Terms

Alzheimer Disease
Animals
Blotting, Western
Caspase 3
Cause of Death
Cell Survival
Computational Biology
Down-Regulation
Flow Cytometry
Humans
Luciferases
Mice*
MicroRNAs
Neurodegenerative Diseases
Neurons*
Plasma
PPAR gamma
RNA, Messenger
Transcription Factor RelA
United States
Up-Regulation
Caspase 3
Luciferases
MicroRNAs
PPAR gamma
RNA, Messenger
Transcription Factor RelA

Figure

• 

  Fig. 1 MiR-128 expression is upregulated and PPAR-γ expression is downregulated in plasma from AD patients and Aβ-treated MCN and N2a cells. (A and B) Expression patterns of miR-128 and PPAR-γ in plasma from healthy volunteers (Normal) (n=20) and AD patients (n=20) were detected using RT-qPCR assay. (C–H) Primary MCN cells and N2a cells were treated with different concentrations of Aβ (0, 5, 10, 20 µM) for 24 h. Then, levels of miR-128 (C and D) and PPAR-γ mRNA (E and F) were determined by RT-qPCR assay, and PPAR-γ protein expression (G and H) was measured using Western blot assay. *p<0.05. PPAR-γ, proliferator-activated receptor gamma; AD, Alzheimer's disease; Aβ, amyloid-β; MCN, mouse cortical neurons; N2a, Neuro2a; mRNA, messenger RNA.

• 

  Fig. 2 Inhibition of miR-128 abates Aβ-mediated cytotoxicity by inactivating NF-κB in MCN and N2a cells. (A–H) MCN and N2a cells were transfected with antimiR-control or anti-miR-128 for 24 h and then treated with different concentrations of Aβ (0, 5, 10, 20 µM) for another 24 h, followed by detection of cell viability (A and B), apoptotic rate (C and D), caspase 3 activity (E and F), and NF-κB activity (G and H). *p<0.05, †p<0.01, ‡p<0.05, §p<0.01. Aβ, amyloid-β; MCN, mouse cortical neurons; N2a, Neuro2a.

• 

  Fig. 3 PPAR-γ is a target of miR-128. (A) Putative binding sites between PPAR-γ 3′UTR and mouse miR-128, and mutant sites in MUT PPAR-γ reporter. (B and C) MCN and N2a cells were co-transfected with WT or MUT PPAR-γ reporter and miR-control or miR-128 mimic for 48 h, followed by measurement of luciferase activity via double luciferase reporter assay. (D–G) MCN and N2a cells were transfected with miR-control, miR-128 mimic, anti-miR-control, or anti-miR-128 for 48 h. Then, PPAR-γ expressions at mRNA (D and E) and protein (F and G) levels were determined by RT-qPCR and Western blot assays, respectively. *p<0.05. PPAR-γ, proliferator-activated receptor gamma; MCN, mouse cortical neurons; N2a, Neuro2a; mRNA, messenger RNA; WT, wild type; MUT, mutant type.

• 

  Fig. 4 PPAR-γ attenuates Aβ-mediated cytotoxicity by inactivating NF-κB in MCN and N2a cells. (A and B) MCN and N2a cells were treated with Control (DMSO), Tro (20 µM), or GW9662 (10 µM) for 24 h, followed by detection of PPAR-γ protein level via Western blot assay. (C–H) MCN and N2a cells were treated with Aβ (10 µM) for 24 h and stimulated with Control (DMSO), Tro (20 µM), or GW9662 (10 µM) for another 24 h. Next, at the indicated time point, cell viability (C and D), apoptotic rate (E and F), and NF-κB activity (G and H) were determined. *p<0.05. PPAR-γ, proliferator-activated receptor gamma; Aβ, amyloid-β; MCN, mouse cortical neurons; N2a, Neuro2a; Tro, troglitazone.

• 

  Fig. 5 miR-128 inhibitor decreases Aβ-mediated cytotoxicity by upregulating PPAR-γ via inactivation of NF-κB signaling in MCN and N2a cells. (A–F) MCN and N2a cells were treated with Aβ (10 µM) for 24 h, followed by stimulation of control (DMSO) or Tro (20 µM) for another 24 h. Aβ-treated cells were transfected with anti-miR-control or anti-miR-128 for another 24 h, together with or without the treatment of control (DMSO) or GW9662 (10 µM) for an additional 24 h. Following this, cell viability (A and B), apoptotic rate (C and D), and NF-κB activity (E and F) were determined in treated cells. *p<0.05. Aβ; amyloid-β PPAR-γ, proliferator-activated receptor gamma; MCN, mouse cortical neurons; N2a, Neuro2a; Tro, troglitazone.

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